Increased post-exercise insulin sensitivity in skeletal muscle is a well-known health benefit of acute exercise, but the underlying mechanisms remain uncertain. The long-range goal is to fully understand the cellular events responsible for this major health benefit. Skeletal muscle is a heterogenous tissue comprised of multiple fiber types with diverse metabolic phenotypes. Conventional tissue analysis cannot discern cellular mechanisms, but a recently developed and validated method enables determination of glucose uptake (GU) and fiber type in single muscle fibers. Recent research using this approach has uncovered striking and unexpected fiber type- selective exercise effects in normal and insulin resistant muscle that were not attributable to lack of recruitment of the fiber types that failed to attain exercise-induced improvement in insulin sensitivity. Akt Substrate of 160 kDa (AS160) is a key insulin signaling protein that regulates GLUT4 glucose transporter translocation. Greater AS160 phosphorylation is consistently linked to greater insulin-mediated GU in whole muscles from normal and insulin-resistant rats. In addition to determination of single fiber GU, this project will use novel methods to measure, for the first time, exercise effects on cellular insulin signaling (including AS160 phosphorylation) and cell surface GLUT4 levels in specific fiber types, thereby advancing understanding from the level of whole muscles to the cellular level of specific fiber types. Newly created AS160-null rats with AAV vector-mediated wildtype or phosphomutated AS160 expression will be used to reveal if AS160 expression or phosphorylation is essential for greater insulin-mediated GU post-exercise in whole muscles and specific fiber types of normal and insulin resistant rats. These unique approaches will make possible unprecedented evaluation of cellular events responsible for the post-exercise increase in insulin sensitivity. The Specific Aims are: 1) To elucidate mechanisms for the exercise-induced improvement in insulin-stimulated GU of whole muscles and specific fiber types from normal rats; 2) To test the mechanisms for high fat diet (HFD)-induced insulin resistance in whole muscles and specific fiber types; and 3) To test the mechanisms for exercise-induced improvement in insulin-stimulated GU of whole muscles and specific fiber types from high fat diet-induced insulin resistant rats. The predicted results are that in whole muscles and fiber types with enhanced insulin-mediated GU after exercise by normal and insulin resistant rats of both sexes, ?3-AMP-activated protein kinase (AMPK) stimulation immediately post-exercise is a trigger that catalyzes greater phosphorylation of AS160 Ser704 (AMPK phosphosite) which acts as a memory element favoring greater insulin-induced AS160 phosphorylation on Thr642 and Ser588, mediators for greater cell surface GLUT4, the end-effector enabling greater insulin- mediated GU post-exercise. We also predict AS160 expression and site-selective phosphorylation are essential for exercise effects on insulin-mediated GU in normal and insulin resistant muscle. This research will provide groundbreaking insights into cellular mechanisms underlying improved insulin sensitivity post-exercise.